PROJECT SUMMARY Genetic mutations cause congenital or progressive inner ear disorders in humans. Despite the recent progress in human genetics for identifying syndromic and nonsyndromic genes, little is known about how mutations in these genes contribute to the clinical features of patients with cochlear and/or vestibular dysfunction. In order to overcome limitations stemming from a paucity of human inner ear tissues available for experimentation, we recently established a method for deriving inner ear sensory epithelia harboring functional sensory hair cells from human pluripotent stem cells in 3D culture. These stem cell-derived tissues, designated as ?human inner ear organoids,? harbor a layer of tightly packed hair cells whose structural, biochemical and functional properties are indistinguishable from native sensory hair cells in the human inner ear. The primary goal of this application is to investigate pathophysiology of two monogenetic inner ear disorders, CHARGE syndrome and DFNA36/DFNB7/11 deafness, with human inner ear organoids as a model system. CHARGE syndrome is a congenital disorder characterized by dysmorphic features of inner ear structures and caused primary by de novo mutations in CHD7, a gene encoding an ATP-dependent chromatin remodeling enzyme. DFNA36 and DFNB7/11 associated maladies are caused by dominant and recessive mutations in TMC1, respectively. Since TMC1 plays a critical role in mechano-electrical transduction of sensory hair cells, hearing loss caused by DFNA36 or DFNB7/11 is believed to originate from defects in sensory transduction. We will generate human embryonic stem cell lines bearing disease-associated mutations using CRISPR/Cas9 genome editing technology, and examine when and how phenotypes manifest themselves using a combination of histological, biochemical and electrophysiological assays. Additional experiments are designed to elucidate the mechanisms underlying the pathological defects and test if some of the defects can be rescued by forced expression of exogenous genes. To our knowledge, this is one of the first studies to recapitulate genetic inner ear disorders using a human model system and will provide valuable clinical information on the etiology of these disorders.